In article <3ti12p$bvi at newsbf02.news.aol.com>, bspinoza at aol.com (BSpinoza)
wrote:
. Consequently, we have expressed
> this protein in the yeast Pichia pastoris.
> 1. Are a lot of people using this system (Invitrogen)?
> 2. What is the experience with improving yields?
> 3. Anyone have any ideas for an interesting collaboration with this
> receptor fragment?
>> Kevin Harris- harrisk at uthscsa.edu
Kevin,
The Pichia system was developed by Philips Petroleum, I believe, as a
single cell food system. They developed these incredibly high yield
fermentations and then it kind of dead ended. Then recombinant proteins
appeared and they knew enough Pichia genetics to create an expression
system. Originally they offered the expression system as a package deal
for $10,000 (or was it $30,000?) where they would sell you the strain, the
vectors and then help you troubleshoot expression. There were few takers.
While the cell density was very high, similar S. Cerv fermentations are
possible with much better systems. There was no guarantee it would work
for your system, either. I think the few takers were companies with big
wallets and small brains, molecular biologically speaking.
Later they gave a license to Invitrogen and the system is reborn. The main
advantage of the system has always been the possibility of very high
density growth and decent secreted protein. This advantage is best
realized with fermentation. Smaller scale cultures are possibly no better
than S. cerv. systems, IMHO. Keep in mind that similar sized
glycosylation does not mean native glycosylation; my experience is that
these homologs tend to be cleared much faster from the blood, albeit not
as fast as ³naked² bacterial proteins, or ³aberrantly glycosylated² insect
proteins.
The typical way to improve Pichia yields is to produce huge cell masses
and then induce via the methanol promoter in a two stage fermentation.
There should be some ref. (included?) from th earlier Philips Pet. work.
Often cystein bridges are the undoing of these fungal systems,
renaturation can help, possibly with disulfide isomerase. Good luck!
Ted Michelini
Institute of Molecular Biology
University of Oregon
Tedm at darkwing.uoregon.edu